Project Summary Histone deacetylase inhibitors (HDACi) are in clinical use in leukemias in combination with agents, such as, chemotherapies. Elucidating their mechanisms of action will lead to mechanism-based combination therapy strategies. Our recently published data suggests a novel mechanism through which HDACi could mediate cytotoxicity. We demonstrate that HDACis differentially acetylate not only NHEJ proteins Ku70/Ku80, but also poly ADP-ribose polymerase-1 (PARP1), known to compete with Ku proteins for binding DSBs. An important finding is that PARP1 binding to chromatin increases with duration of HDACis exposure, resembling PARP ?trapping? recently demonstrated with PARP inhibitors. PARP1 knockdown inhibits chromatin trapping and mitigates HDACis effect on NHEJ. Moreover, HDACis combined with the potent PARP inhibitor (PARPi) BMN673 induce a dose-dependent increase in PARP trapping. These results provide a new mechanism by which HDACis both acetylate PARP1 and increase binding of PARP1 to DSBs, leading to decreased access of C-NHEJ factors to DNA damage sites, decreasing repair of cytotoxic DSBs in leukemia cells. Validation of this novel concept through the studies we propose, will provide a compelling mechanism-based approach for combining HDACis with PARPis to enhance cell cytotoxicity in acute leukemias. In this proposal we will focus on acute myeloid leukemia (AML) for which novel therapies are needed, to test the central hypothesis that clinically relevant HDACis can cause cytotoxic DSBs through acetylation of PARP1, leading to PARP trapping in chromatin. HDACis in combination with PARPis will enhance PARP trapping in AML cells leading to increased cell cytotoxicity. In specific aim 1, we will determine the extent to which acetylation of PARP1 induced by HDACis contributes to PARP trapping and decreased NHEJ activity. To elucidate the precise molecular steps leading to PARP trapping in chromatin and decreased NHEJ activity, we will use ?state of the art? assays and study clinically- relevant HDACis in AML cell lines stably knocked down for PARP1 and the effects of over-expressing PARP1 mutants of 3 key lysine residue targets of acetylation. In specific aim 2, we will determine the sensitivity of HDACis in combination with PARPis in primary AML cells in vitro and in vivo. We will use ?state of the art? primary mouse models to study the effects of these inhibitors. These studies will elucidate new mechanisms involving HDACis that will lead to future mechanism-based clinical strategies to combine HDACis with PARPis as a therapy approach in leukemias and other cancers.